Ecdysis sequences are stereotyped motor programs that are used to cast off an insects exoskeleton at each molt and to expand a new exoskeleton to accommodate further growth. Survival and growth thus depend upon successful execution of ecdysis sequences, and each such sequence must be tailored to the animals particular developmental stage. The developmental stage that has been the focus of most of the research in my laboratory is that of the newly metamorphosed adult, where the ecdysis sequence includes an adaptive, environmentally-sensitive behavioral program that mediates the search for suitable surroundings, and a subsequent, hormonally-driven program that serves to expand the recently developed wings of the newly emerged fly. As summarized in an invited review published this year in the Annual Reviews of Entomology (White & Ewer, 2014, Annu. Rev. Entomol. 59: 363-381), the first program is used to find a safe perch from which the immobile fly can expand its wings, and the second program initiates expansion. Because wing expansion must be undertaken within several hours of emergence, the need for safety must be balanced by the imperative to expand and each individual fly must decide when (and under what environmental circumstances) to expand. Wing expansion thus provides a behavioral paradigm for studying decision-making, the most fundamental aspect of behavioral integration, and for understanding how hormonal and environmental factors act, individually and in concert, to recruit motor patterns to assemble behavioral sequences. Such understanding should, in turn, shed light on the deficits in behavioral organization that lie at the root of many mental disorders, including obsessive-compulsive disorder, schizophrenia, and bipolar disorder. My laboratory's approach to understanding how the Drosophila nervous system produces behavioral sequences crucially depends on genetic techniques that allow specific subsets of brain cells to be turned off or on in freely behaving animals. This approach requires genetic tools for both suppressing and stimulating brain cell activity, as well as tools to target these manipulations to the desired subset of cells. Making such tools is another principal goal of our research. Two tools that have driven much of our recent research include a technique for acute neuronal activation using the mammalian cold-sensitive channel TRPM8, which we introduced in 2009 (Peabody et al., 2009, J Neurosci. 29:3343-53), and a technique for targeting transgene expression to neurons that express a specific gene of interest, which we introduced in 2012 (Diao & White, 2012, Genetics 190:1139-44). The ability to acutely activate neurons using TRPM8 was central to a project carried out in collaboration with the laboratory of Dr. Moto Yoshihara in which the goal was to develop a general method for identifying core circuit components responsible for generating simple behaviors in the fly. A manuscript describing this method was published during the reporting period in the journal G3 (Flood et al., 2013, G3:Genes, Genomes, Genetics 3:1629-37, and one of the principal successes of applying the approach was described in an earlier publication in Nature, which characterized a pair of command neurons that controls feeding behavior in Drosophila (Flood et al. , 2013, Nature 499:83-7). We have also used acute activation by TRPM8 to characterize the command neurons for wing expansion behavior, which we identified previously (Luan et al., 2012, J Neurosci 32: 880889). The recent work, published in the Journal of Experimental Biology (Peabody & White, 2013, J. Exp. Biol. 216:4395-4402), shows that the command neurons become competent to drive the wing expansion program only after emergence from the pupal case. Prior to this time the wing expansion network is suppressed--likely downstream of the command neurons--and the act of emergence, or some process closely associated with it, lifts this suppression. Our results provide insight into how nervous systems use inhibitory mechanisms to assemble motor programs into correctly ordered behavioral sequences. The T2A-Gal4 in-frame fusion technique couples the expression of transgenes to the expression of endogenous genes of interest using the ribosomal skipping mechanism of the viral T2A peptide. We had previously used this technique to make a fly line that co-expresses the Gal4 transcription factor in cells that express the receptor for bursicon, the hormone that governs wing expansion. In addition to using this line to characterize the functional roles of bursicon receptor-expressing neurons in the wing expansion, we also identified a novel set of cells in the fly gut that use bursicon signaling to regulate stem cell turnover. This work was carried out in collaboration with the laboratory of Dr. Marcos Vidal and was recently published in the journal Current Biology (Scopelliti et al., 2014, Curr. Biol. 24: 1199-1211). A second manuscript, in which the T2A technique was exploited to characterize the circuitry underlying color processing in the Drosophila brain, was the product of a collaboration with the laboratory of Dr. Chi-hon Lee and was published in the journal Neuron. (Karuppudurai et al., 2014, Neuron 81: 603-615). In addition to exploiting the T2A-Gal4 in-frame fusion technique to investigate specific biological questions, we have also spent considerable effort over the last two years further developing this technique. To extend its range of application, we have developed a broad toolkit of plasmids and fly lines that permits the technology to be easily used in conjunction with the MiMIC transposon-containing lines produced by the Drosophila Gene Disruption Project. Our MiMIC-T2A-mediated In-Frame Fusion method (i.e. MiMIC-TIFF) allows Gal4 and numerous other transgenes to be expressed in the same pattern as endogenous genes that contain MiMIC insertions. The method works for any MiMIC insertion that lies within the coding intron of a gene of interest and uses recombinase-mediated cassette exchange to replace the MiMIC insert with a T2A fusion construct preceded by a splice acceptor site. Currently, there are several thousand suitable MiMIC insertions available for use with this technique, and we have successfully tested the technique with T2A constructs that permit expression of not only Gal4, but also Gal80 and components of the Split Gal4 system, which we introduced in 2006 (Luan et al., 2006, Neuron, 52:425-36). A manuscript describing this technique is expected to be submitted later this year. A second manuscript in preparation describes the application of the MiMIC-TIFF technique to characterize neurons that express the receptor of Ecdysis Triggering Hormone, which is the master regulator of ecdysis sequences in flies and other insects. In summary, we have made substantial progress during the last year in elucidating, or helping to elucidate, several behavioral circuits in the Drosophila nervous system, including the one that supports wing expansion. At the same time, we have continued to develop tools that will support not only our own circuit mapping efforts, but also those of other members of the Drosophila research community. As we use these tools to extend and refine our analysis of the wing expansion circuit andmore broadly, the circuit that underlies ecdysis sequences at all developmental stagesour work should provide insight into the principles used by all nervous systems to generate and organize behavior